Adenosine 5'-triphosphate binding to bovine serum albumin.
Binding of ATP to bovine serum albumin was shown by ultrafiltration and NMR. The binding was pH dependent. Scatchard analysis revealed that at pH 5.4, 6.4 and 7.4, dissociation constant Kd was 13, 40 and 120 microM, respectively, and no binding was observed at pH 8.4. The binding stoichiometry was 1:1 for all pH. Dimer of BSA did not bind ATP. From chemical shifts of 31P-NMR, Kd was estimated to be 15 microM at pH 5.4, which is very close to that determined by ultrafiltration. While adenosine did not interfere with the binding. GTP, dCTP, ADP, UTP, AMP, phosphate and pyrophosphate were competitive inhibitors and their inhibition constants Ki were 25, 32, 36, 50, 130, 1000 and 186 microM, respectively. Fatty acids such as lauric acid and palmitic acid did not interfere with the binding. Warfarin was a non-competitive inhibitor. Cl- competitively inhibited the binding, and the inhibition constant was 20 mM. The dissociation constants of the Cl- binding were reported to be 0.42 mM for the first binding site, 10-5 mM for the second and 303-143 mM for the third [G. Scatchard, W.T. Yap, J. Am. Chem. Soc., 86 (1964) 3434; G. Scatchard et al., J. Am. Chem. Soc. 79 (1957) 12]. This suggests that the ATP binding site may be the second Cl- binding site.
Takeda
S
S
Laboratory of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Miyauchi
S
S
Nakayama
H
H
Kamo
N
N
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D019281:Dimerization; D011756:Diphosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D014462:Ultrafiltration; D014859:Warfarin / Q000494:pharmacology
D002712:Chlorides; D011756:Diphosphates; D010710:Phosphates; D012710:Serum Albumin, Bovine; D014859:Warfarin; D000255:Adenosine Triphosphate